Institut Sophia Agrobiotech

Legume-Rhizobium Symbiosis: cellular redox status

The team focuses on improving our knowledge of plant/nitrogen-fixing bacteria (Rhizobium) by studying the role of the cellular redox state during the symbiotic interaction.

Background, Research topics and objectives

The growth of cultivated plants largely depends on the presence of a sufficient nitrogen quantity in soil. The massive use of nitrogen fertilizers in intensive farming is a considerable risk for the environment, leading to major pollution of the environment by nitrate and nitrite. Among plant-microbe interactions, the symbiosis between nitrogen-fixing rhizobia and legumes represent a major opportunity for the rational management of nitrogen inputs in agriculture and for the reduction of the soil pollution. During symbiosis, essential cellular functions of plant cell are manipulated by the symbiotic bacteria in order to allow its penetration. This leads to the formation of a new organ, the so-called nodule, capable of fixing atmospheric nitrogen and thus allowing the plant to meet most of its nitrogen requirements. However, this symbiosis is very sensitive to environmental changes and its lifespan is limited: the developmental nodule senescence, a breakdown of the symbiotic association, appears before the plant senescence. Thus, one major characteristic of the relationship between both partners is a switching from a compatible to an incompatible interaction.The detailed understanding of mechanisms involved in the establishment and the maintaining of this benefic symbiotic relation is essential for the development of strategies to improve plant health and contribute to more environmentally-friendly agricultural practices.

Using the Medicago truncatula/Sinorhizobium meliloti interaction as symbiotic model, the research conducted in our team aims at studying the role of the cellular redox state in this interaction and deciphering mechanisms leading to the nodule senescence in both partners. Within this context, the roles of glutathion (GSH), hydrogen peroxide (H2O2) and nitrogen monoxide (NO), three major molecules involved in the regulation of the cellular redox state, are analyzed. In parallel, the importance of plant cysteine protease and bacterial toxin-antitoxin systems is investigated during nodule senescence

Biological models studied

Scientific expertise

The team has long-standing experience of the nitrogen fixation process and of the involvement of reactive oxygen species in it. However, very few data exist and few studies have been devoted to the importance of the cellular redox state in symbiotic legume/bacteria interactions. Moreover, nodule senescence (aging) is a mechanism that has received little attention and is therefore not yet well understood.

Current priorities

What is the role of the molecules involved in the regulation of the cellular redox state (H2O2, NO, GSH) in nodule establishment, function and senescence? More specifically:What is the role of H2O2 in the establishment and function of symbiosis (molecular target, origin and dynamics of H2O2 production, etc.)?What are the mechanisms regulated by GSH and NO in the formation and maintenance of nodules?What is the contribution of the bacteroid to nodule senescence?

Fig. 2 : Study of symbiotic Rhizobium-legume interactionE. Visualization of Bacteroids by electron microscopy during salt stress. F. Study of the expression of ferritin in a salt stress by in situ hybridization. G. Characterization of gene expression and synthesis of glutathione homoglutathion by northern blot.

Application

To reduce the quantity of nitrogen fertiliser used on crops by improving the natural fixation of atmospheric nitrogen produced through symbiosis.

Major results

Development of new cellular tools to analyze the symbiotic nitrogen fixing interaction in vivo.

In order to better understand the nitrogen fixing symbiosis, we have developed new cellular tools. In this context, multiple probes analyzed using confocal microscope imaging were developed and used in nitrogen fixing nodules. PBS pH was measured in vitro during the whole symbiotic process using ratiometric fluorescent probe. This analysis showed that nitrogen fixing zone maturation goes with the acidification of the peribacteroid space in the nitrogen-fixing organite, the symbiosome (Pierre et al., 2013). The viability of bacteroids was analyzed during their differentiation and throughout the symbiotic interaction in vivousing nodule sections with the Live/Dead® BacLightTM probe. Finally, the in vivo quantification of H2O2 was measured in nodule using a protein probe HyPer. This analysis correlates the production of H2O2 with the regulation of MtSpk1, an H2O2 regulated gene (Andrio et al., 2013).

Identification and characterization of H2O2 and NO-regulated genes in the rhizobial symbiosis.

Reactive oxygen species (ROS), particularly H2O2, and nitric oxide (NO) play an important role in signalling in various cellular processes. The involvement of these molecules in the Medicago truncatula-Sinorhizobium meliloti symbiotic interaction raises questions about their effect on gene expression. Transcriptomic analyses were performed on inoculated roots of M. truncatula to identify genes regulated by these two molecules. Numerous genes were found to be differential regulated in H2O2 and NO-depleted roots during the symbiotic interaction. Characterisation of candidate genes such as MtSpk1, a putative protein kinase, showed their involvement in the signal transduction pathway involved in the symbiotic interaction (Andrio et al., 2013 ; Boscari et al. 2013).

Mandon K. MCU since 1997 since September 2012 . Coordinator Diploma Bachelor's Degree in Life Sciences from the University of Nice- Sophia Antipolis (750 students). Since September 2012: Coordinator of the course " Molecular Biology and Genetics A- Pet ; B- Plant " 3rd year of License SVS (30 students) since September 2006 . Responsible for Plant Physiology Teaching team .